dr graham ogg mrc programme leader, oxford consultant dermatologist dermatological danger
TRANSCRIPT
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Dr Graham OggMRC Programme Leader, Oxford
Consultant Dermatologist
Dermatological Danger
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Cutaneous inflammatory patterns
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exogenous antigens eg atopic
endogenous antigens eg varicella zoster virus
Aim: To understand role of human cutaneous T cells in mechanisms of disease, treatment and vaccination
HLA class I HLA class II
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T cell recognises antigen presented by HLA class I/II
CD4/CD8TCR
HLA
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HLA class II comprises 2 chains
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T cell receptor and HLA class II
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HLA class II
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How does the antigenic peptide get to HLA class II?
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Endosomes fuse with vesicles containing proteolytic enzymes
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These fuse with vesicles containing receptive HLA class II
HLA class II
Invariant chain
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Each HLA class II binds peptides carrying preferred motifs
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Th2 vs Th1
IFN productionCD8+ T cell helpMacrophage activationIgG class switching
IL-4 productionIgE class switchingEosinophil recruitment
CD4+ T cell recognition of target cell leads to:
1. Cytokine production2. Proliferation of T cell (clonal expansion)
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HLA class I
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T cell receptor/HLA class I
T cell receptor
MHC class I
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HLA Class I (T cell receptor view)
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HLA class I antigen presentation
proteasome
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Some degraded peptides enter the endoplasmic reticulum
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CD8+ T cell recognition of target cell leads to:
1. Lysis of target cell2. Cytokine production3. Proliferation of T cell (clonal expansion)
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pep 4negativecontrol
pep 12
ELISpot can be used to detect cytokine secreting cells
positivecontrol
Bateman et al JACI 2006
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HLA-peptide tetrameric complexes
Ogg et al Science 1998Champagne/Ogg et al Nature 2001Seneviratne et al J Clin Invest 2002
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HLA tetramers allow us to look at T cells that are specific for a particular antigen
Blood Tissue
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HLA class I HLA class II
KeratinocytesFibroblastsMelanocytesOthers
Langerhans cellsDermal dendritic cellsKeratinocytes (under inflamm conditions)
Cells in the skin that might present antigen to T cells
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Atopic dermatitis (eczema)
•Cumulative prevalence up to 15-20%
•Onset usually by age 2-6 months
•50-75% of children clear by age 10 years
•50% have associated asthma and/or hayfever
•Staphylococcus aureus presence common (cf impetigo)
•80% have IgE and/or skin test reactivity to common environmental allergens
•FLG null mutations common
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•Genome screens detected linkage to eg 3q21, 1q21, 17q25 and 20p (similar to psoriatic susceptibility loci). Numerous candidate gene analyses eg FcRI, IL-4, IL-10, IL-13, SPINK5, TLR2.
•Null mutations in FLG are commonly associated with atopic dermatitis
Atopic dermatitis – genetics and environment
Palmer et al Nature Genetics 2006
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Filaggrin expression is variable and is inhibited by Th2 cytokines
Howell et al JACI 2007
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Severe atopic dermatitis is associated with common FLG null mutations in our cohort
Cohort 2282del4
hetero
2282del4
homo
R501X
hetero
R501X
homo
Total >1 null mut
32 3 0 3 2 8
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Working model of disease
Barrier
Allergen Infection
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Individuals with atopic dermatitis have high frequencies of circulating allergen-specific Th2 cells
Der p 1 peptides
Non-atopics
Atopics
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Ex vivo
Allergen-specific CD4+ T cells proliferate in vitro
Cultured ELISpot
Ardern-Jones et al 2007 PNAS
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T cell epitope hunting
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HLA-peptide tetrameric complexes
Ogg et al Science 1998Champagne/Ogg et al Nature 2001Seneviratne et al J Clin Invest 2002
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1.65%
2.34%
0.29%
5.3% 0.02%
0.01%0.54%
0.44%
PATIENTS CONTROLS
CD4
Tetramer
AD5
AD6
AD10
AD22
AD25
AD18
AD9
AD14
A
0.03%
N
J
A
0.02%19.13%
9.9%
AD controls0.001
0.01
0.1
1
10
100 P<0.05
Perc
enta
ge C
D3+
and
Tetr
amer
+
Individuals with atopic dermatitis have higher frequencies of circulating Der p 1-specific CD4+ T cells than non-atopics
(short term culture)
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What about other forms of barrier compromise
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Wasp venom specific T cells responses
Aslam et al Clin Exp Allergy 2006
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•Hyaluronidase
•Antigen V
•Phospholipase
Dominant T cell antigens within wasp venom are co-incident with main IgE binding proteins
Aslam et al CEA 2006
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Mapping Ves V5 epitopes
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Antigen-specific CD4+ T cells infiltrate skin after antigen challenge
10%0.04%
PBMC Skin
DRB1*1501/IE63 tetrameric complex
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CD80
HLA-DR
CD86
CD56
HLA-ABC ICAM-1
Solid line = untreatedDashed line = overnight with IFN-
IFN increases class I, class II and ICAM-1 expression by keratinocytes
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IFN treated keratinocytes can engulf fluorescent latex particles
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IFN treated keratinocytes can present antigen to CD4+ T cells using either peptide or recombinant protein
Black et al EJI 2007
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Keratinocyte killingCell count 040506
Nil 1:1 10:1 40:10
250
500
750
1000Clone 1Clone 8Clone 10Clone 14
Nu
mb
er /
10x
fie
ld
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Increase in number of IL-4-producing T cells using combined stimulation of Der p 1-specific line with
peptide and Staphylococcal enterotoxin Bsf
u/40
,000
cel
ls
stimulus
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Supernatant from SEB/PBMC enhances antigen presenting capacity of keratinocytes
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IFN within supernatant of SEB-treated PBMC enhances class II and ICAM-1 expression by keratinocytes and
enhances presentation to allergen-specific CD4+ T cells
Ardern-Jones et al
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Depletion of IFN from supernatant of SEB+PBMC diminishes ability of supernatant to promote keratinocyte presentation of
peptide
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IL-4 depletion significantly reduces the production of cytokines by allergen-specific CD4+ T cells
Ardern-Jones et al
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SEB
SEB-reactiveT cell
Allergen-specificT cell
IL-4
IFN-
SEB-reactive T cells produce IFN and IL-4 which enhances responsiveness of allergen-specific T cells
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Conclusions
• FLG mutations associate with increased circulating airborne allergen-specific Th2 cells.• FLG mutations do not associate with circulating wasp venom-specific Th2 cells.• These suggest that barrier factors plus Th2 susceptibility important for allergic responses.• Keratinocytes can promote Th2 responses• Antigen-specific CD4+ T cells can infiltrate skin• Combined presence of S.aureus and allergen enhances allergic inflammation.
• Handling of concurrent adjuvant is likely to be an important co-factor in determining Th1/Th2 response to a given antigen
• MRC Experimental Medicine proof of concept clinical trial
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Acknowledgements
Louise Jones
Neelika Malavige
Antony Black
Tess McPherson
Aamir Aslam
Michael Ardern-Jones
Laura Crack
Hsien Chan
Carol Hlela
Elizabeth Bateman
Funding
MRC
NIHR
Milica Vukmanovic-StejicArne Akbar
UCL